The present invention relates to a method for producing hybridisation complexes whose stability is substantially independent of the base composition of two hybridised nucleic acid molecules. The invention also relates to the use of this method for the assay of nucleic acid sequences. The invention further relates to hybridisation complexes produced from two complementary, or substantially complementary, nucleic acid molecules whose stability is substantially independent of the base composition of said two hybridised nucleic acid molecules.
The pairing law A-T (or A-U) and G-C gives nucleic acids the property of forming specific hybridisation complexes between complementary sequences. This long known hybridisation property means that a fragment of nucleic acid, or an oligonucleotide, can be used as a probe to display the presence of a complementary nucleic sequence. The analysis of DNA fragments after gel separation (E. Southern, J. Mol. Biol., 1975, 98, 503-517) is today widely used both in the area of fundamental research and in medical analysis (Caskey, Science, 1987, 236, 1223-1228; Landegrer et al., Science, 1988, 242, 229-237; Arnheim et al., Ann rev. Biochem., 1992, 61, 131-156).
Several assay techniques of nucleic acids based on their hybridisation property with probes have been developed, in particular for the sequencing of unknown DNAs or RNAs, the detection of sequences associated with a pathology, and the search for point mutations in sequences (S. Ikata et al., Nuclei Acids Res., 1987, 15, 797-811; J. A. Matthews, L. J. Krieka, Analytical Biochemistry, 1988, 169, 1-25).
More recently, new assay and sequencing methods for unknown DNA fragments have been put forward, based on hybridisation with a series of oligonucleotides immobilised on a solid support (E. Southern, European Patent published under number: 0 373 203; K. R. Khrapko et al., FEBS Letters, 1989, 256, 118-122; R. Drmanac et al., Genomics, 1989, 4, 114-128; R. Drmanac et al., DNA and Cell Biology, 1990, 9, 527-534; K. R. Khrapko et al., J. DNA Sequencing Mapp., 1991, 1, 375-388; R. Drmanac et al., Science 1993, 260, 1649-1652; R. J. Lipshutz, J. Biomol. Struct. Dyn., 1993, 11, 637-653; U. Maskos, E. Southern, Nucleic Acids Res., 1993, 21, 4663-4669; A. C. Pease et al., Proc. Natl. Acad. Sci., USA, 1994, 91, 5022-5026; J. C. Williams, Nucleic Acids Res., 1994, 22, 1365-1367; E. M. Southern, Nucleic Acids Res., 1994, 22, 1368-1373. These new methods consist of detecting the signals emitted by hybrids produced by pairing a labelled DNA with a series of oligonucleotides having the same length and different sequences immobilised on a membrane or glass surface, then of reconstructing the sequence of the DNA fragment using an algorithmic process. The assay of the hybridisation complexes obtained with the above methods is based on the greater stability of the hybrids without mismatch in relation to that of hybrids containing one or more mismatches. The distinction between a perfect hybrid and a hybrid with mismatch may be made for example by raising the temperature of the medium, which produces dissociation of the complexes with mismatch before dissociation of the complexes without mismatch.
However, the use of natural oligonucleotides in the preceding techniques which also use a great number of these probe oligonucleotides, comes up against a fundamental difficulty connected with the difference in hybrid stability in relation to the base composition of the immobilised oligonucleotides. Indeed, it is known that the base pair G-C, characterized by three hydrogen bonds, is more stable than the pairs A-T or A-U which only have two hydrogen bonds. Therefore, the stability of perfect hybrids produced by sequences with a high number of A-T pairs, is likely to be close to or even less than the stability of imperfect hybrids comprising a mismatch and produced by sequences having the same length but with a high number of G-C pairs. This phenomenon leads to false-positive or false-negative signals depending upon hybridisation temperature and wash conditions.
The use of oligonucleotides formed by the natural nucleosides dA, dG, dC, T or dU, therefore raises a major obstacle against the development of these new techniques for the assay of nucleic acid sequences. The risk of failure in distinguishing between perfect and imperfect hybrids also implies systematic control of a great number of oligonucleotides under determined temperature and medium conditions.
To remedy this drawback, the prior art proposed a hybridisation method using tetramethylammonium chloride to reduce differences in hybrid stability related to base composition (W. B. Melchior, P. H. von Hippel, Proc. Natl. Acad. Sci., USA, 1973, 70, 298-302; U. Maskos, E. M. Southern, Nucleic Acids Res., 1993, 21, 4663-4669), but the results obtained were contradicted by other nucleotide sequences (P. V. Riccelli, A. S. Beright, Nucleic Acids Res., 1993, 21, 3785-3788).
The suggestion was also put forward of varying the density of the oligonucleotides immobilised on polyacrylamide gel in relation to their base composition (K. R. Khrapko et al., J. DNA Sequencing Mapp., 1991, 1, 375-388). But this strategy considerably complicates the system when it is applied with a great number of immobilised oligonucleotides. The use has also been contemplated of modified bases such as 5-CldU and 2-NH2dA, however substantial variation in hybrid stability was observed (J. D. Heisel, H. Lehrach, FEBS Lett., 1990, 274, 103-106). Also, European patent application EP-A-0 176 396 describes a method for producing a complex between a nucleotide probe and a nucleic acid in which at least one part of the adenine groups of the probe is replaced by groups of modified adenine. The modified adenine groups are likely to form three hydrogen bonds and therefore increase the stability of the complex produced. The gain in stability obtained with such probes enables hybridisation to be produced despite the presence of mismatches.
It was also suggested, in international patent N• WO-A-305 175, to make use of nucleotides comprising analogues of pyrimidic bases in PCR or hybridisation reactions.
The bases used in the methods described in D2 form pairs whose stability is close both to A and to G.
Another approach using oligomers constructed with deoxyribonucleosides and ribonucleosides to destabilise the hybrids of sequences with a high number of G and C bases has also been described (Jörg D. Hoheisel, Nucleic Acids Research, 1996, vol. 24. No. 3), but the quantity of hybrids produced with these oligonucleotides falls very rapidly when the alternation number increases. Also, heat dissociation of these hybrids extends over a very wide temperature zone, which means that differentiating between perfect hybrids and hybrids with mismatch becomes largely unreliable.